Summary: The dengue virus (DEN) protease is a heterodimer of nonstructural protein 2B (NS2B) and NS3. Previously, we performed a mutagenesis study of some of the residues in NS3 of DEN type 2 (DEN2) predicted to be involved in substrate binding. In all, 46 mutations were analyzed for their effect on cleavage in vitro. Thirteen of these mutants with wild-type or nearly wild-type activity were analyzed by reverse engineering into a full-length DEN2 cDNA clone and testing mutant RNA transcripts for infectivity: 6 were non-infectious, while 7 were recovered as virus. The recovered viruses had wild-type growth kinetics. Now, 8 members of a previously-made set of mutations in the region of DEN4 NS2B essential for protease activity have been engineered into an infectious DEN4 clone made by Robin Levis. Virus was recovered from 5 of these, while 3 were lethal. One of the recovered viruses was a partial revertant. Several of the viruses had a second-site mutation at another location in NS2B, changing a specific met residue to ile or val, complicating the analysis of the the effect of the introduced mutations on the phenotype. One of the recovered viruses had a significant growth defect. We had previously observed that transcripts made from DEN2 cDNA clones with short deletions at the 3' end are infectious. Constructs missing up to 6 nt are viable, while deletion of 8 or more nt is lethal. Deletions of 7 nt are viable or lethal, depending on the presence and composition of a restriction site overhang at the 3' end of the run-off transcript. In all but one case, the 3' end sequences of the recovered viruses are wild-type; virus recovered from the 7 nt deletion has a 3' end sequence that differs from wild-type at 1.5 positions, but this mutant virus appears to grow with wild-type kinetics. To investigate this further, we have continued our collaboration with the Padmanabhan lab, now located at Georgetown. Several small 3' end deletions are being analyzed in their in vitro RNA replication system. So far, it is clear that the deleted RNAs are replicating, and analysis of the 3' end sequences of replicating RNAs is ongoing. We have two other ongoing collaborations with the Padmanabhan group. (1) Several double mutants in DEN2 NS3 were constructed, and are being expressed in E coli and purified, for subsequent crystallographic analysis. We are also trying to express and purify a dimer between NS3 and the protease domain of NS2B. (2) We have attempted to make a DEN2 replicon expressing green fluorescent protein (GFP). The initial results do not look promising, in that the level of GFP expression is very poor. Work is underway to change transgenes. Finally, in a side project, a collaboration was done with Natasha Caplen and Richard Morgan at the NIH, demonstrating the inhibitory effect of small dsRNA on gene expression and on the growth of dengue virus in C6/36 mosquito cells. This has resulted in a publication.